Abstract
Pyruvate, orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthetase (PEPS) catalyze the conversion of pyruvate to phosphoenolpyruvate (PEP). Both are regulated by a phosphorylation-dephosphorylation mechanism involving a bifunctional serine/ threonine kinase and a pyrophosphorylase (PPDK regulatory protein, PDRP, and PEPS regulatory protein, PSRP, respectively). In plants the regulatory mechanism involves phosphorylation of a threonine residue that is separated by a single amino acid from the histidine residue that forms a phosphorylated intermediate during catalysis. Using antibodies, we demonstrated that the regulation of both Listeria monocytogenes PPDK and Escherichia coli PEP synthetase involves the phosphorylation of a threonine residue located close to the catalytic histidine residue. The amino acid located between the regulatory threonine and the catalytic histidine is highly conserved being serine in PPDK and cysteine in PEPS. Using site-directed mutagenesis we have shown that both PPDK and PEPS in which the serine and cysteine residues, respectively, were substituted with an alanine the enzymes could be regulated indicating that the serine and cysteine residues, respectively, are not essential for regulation. We also demonstrated that altering the intermediate amino acid did not alter the specificity of the regulatory proteins for their protein substrates.
Highlights
Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1) catalyzes the reversible conversion of pyruvate to phosphoenolpyruvate (PEP) and is important in C4 plants as it is generally recognized as catalyzing the rate-limiting step of C4 photosynthesis [1]
We demonstrated that the regulation of both Listeria monocytogenes PPDK and Escherichia coli PEP synthetase involves the phosphorylation of a threonine residue located close to the catalytic histidine residue
The PPDK regulatory protein (PDRP) from L. monocytogenes was successfully expressed at 16 ̊C and was soluble and stable when stored on ice
Summary
Orthophosphate dikinase (PPDK; EC 2.7.9.1) catalyzes the reversible conversion of pyruvate to phosphoenolpyruvate (PEP) and is important in C4 plants as it is generally recognized as catalyzing the rate-limiting step of C4 photosynthesis [1]. The enzyme is regulated by three separate mechanisms; by light-dependent gene expression [2,3], end-product inhibition [4] and by lightdependent post-translational modification [5]. The lightdependent post-translational regulation of PPDK is unusual in that it requires the protein to be catalytically phosphorylated prior to inactivation [6], involves an ADP-dependent phosphorylation [7], the activation involves a Pi-dependent dephosphorylation that results in the production of PPi [7] and, a single enzyme catalyzes both the inactivation and the activation reactions [8]. The cloning of maize PPDK regulatory protein (PDRP) revealed the regulatory protein had been sequenced from more than 200 bacterial species and had been identified as DUF299 (domain of unknown function).
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